Contrary to our prediction, Experiment 1 showed that increasing the duration of the ethanol-free interval between periods of passive IG ethanol exposure from 10–12 h (Massed Group) to 34–36 h (Spaced Group) significantly reduced the amount of ethanol self-infused during subsequent choice days (). Thus, even though they were exposed to significantly more ethanol during the passive phase, the Spaced Group self-infused less ethanol than the Massed Group during the choice phase. Moreover, Spaced Group rats developed a significant aversion for the S+ flavor whereas Massed Group rats did not. Examination of drinking patterns showed that the group difference in self-infused ethanol could be explained by a difference in the daily number of ethanol bouts rather than by a difference in ethanol bout size. Experiment 2, which failed to show an effect of the time delay (12 vs. 36 h) between the last passive ethanol exposure and onset of self-infusion (), suggested that the greater self-infusion by Massed Group rats in Experiment 1was not due to use of the shorter time delay for that group. Finally, Experiment 3 showed that inserting no-choice self-infusion days between the last few passive exposure days did not affect subsequent self-infusion ().
With the exception of the Spaced Group, the daily amounts of ethanol self-infused by rats that received our new passive phase dosing regimen (group means ranging from 4.1 to 7.9 g/kg/d) were as high or higher than those shown by rats that were passively exposed to ethanol using the temperature-feedback protocol described in our earlier studies (group means ranging from 3.8 to 4.9 g/kg/d, Experiments 1–3; Fidler et al., 2006
). It is notable that all of these passive infusion paradigms were intermittent at least to some extent. Groups exposed to the Massed procedure in the three experiments reported here received three passive ethanol infusions per day (with infusions starting 340 min apart) followed by an ethanol-free period of at least 10 h. In contrast to the high intakes produced by exposure to the Massed procedure, the low daily amount of ethanol self-administered by the Spaced Group during the choice phase (2.2 g/kg/d) was quite similar to the levels previously observed in four groups of control rats that were infused with water or nothing during the passive phase (group means ranging from 1.0 to 2.2 g/kg/d, Control Experiment; Fidler et al., 2006
). The Spaced Group was also similar to previous control groups in showing fewer ethanol bouts per day than the Massed Group. Although such cross-experiment comparisons must be interpreted cautiously, our findings in the Spaced Group suggest that lengthening the time delay between each series of passive infusions to 36 h largely eliminated the enhancing effect of passive ethanol exposure on later IG self-infusion. It is not known whether similar increases in the “off” time in an intermittent ethanol vapor exposure protocol (e.g., 16 hours on/8 hours off, Lopez & Becker, 2005
) would reduce or eliminate the enhancing effect of chronic vapor exposure on ethanol drinking or operant oral self-administration. However, this outcome seems likely in light of earlier data showing that extending the time interval between consecutive periods of ethanol vapor exposure to 24 h eliminated the dependence enhancing effects of repeated vapor exposure seen at shorter time intervals (Goldstein, 1974
). As it stands, the ethanol exposure induced by an ethanol vapor protocol is quite different from that induced in our passive infusion protocol. As far as we are aware, no studies have been done where ethanol vapor is delivered in brief exposures that would mimic the IG procedure reported here.
Examination of the intoxication and withdrawal scores suggests at least two possible interpretations of the self-infusion difference between the Massed and Spaced groups. One possibility, suggested by the finding that the groups had similar intoxication scores despite the higher level of passive ethanol exposure in the Spaced Group, is that the Spaced Group developed greater tolerance to ethanol-induced impairment than the Massed Group (i.e., a higher ethanol dose was needed to produce the target level of impairment in the Spaced Group). This interpretation might explain the lower self-infusion in the Spaced Group if one assumed that tolerance to ethanol’s impairing effect was accompanied by near complete tolerance to its reinforcing effect. However, given the paucity of data showing development of tolerance to ethanol reinforcement or data showing that more widely spaced ethanol exposures enhance ethanol tolerance, this interpretation is not compelling. It seems more likely that the greater sensitivity of the Massed Group to the impairing effects of ethanol was due to their shorter period of recovery after each set of passive ethanol infusions. That is, the impairing effects of ethanol may have combined additively with the general fatigue produced by each series of three closely spaced ethanol infusions in the Massed Group.
Our withdrawal data offer stronger support for an alternative interpretation of the Massed-Spaced self-infusion difference based on differences in development of ethanol dependence (with degree of dependence inferred from the magnitude of the withdrawal response). More specifically, the Massed Group showed significantly higher withdrawal scores than the Spaced Group 12 h after onset of the last daily infusion (). Moreover, rats exposed to a Massed procedure in Experiment 2 (Delay Group) showed much stronger withdrawal 30–36 h after the last passive infusion than Spaced Group rats tested at the same long post-infusion delays in Experiment 1. Both observations support the general conclusion that the Massed procedure induced a greater degree of dependence than the Spaced procedure. Thus, the higher level of choice ethanol self-infusion produced by the Massed procedure might be explained by the opportunity for greater negative reinforcement by ethanol (i.e., greater alleviation of aversive effects of ethanol withdrawal). Given the temporal pattern of withdrawal over time after the Massed procedure, it is possible that the ability of passive exposure to enhance subsequent self-infusion depends on having access to ethanol during the time period between 24–48 h after the last passive infusion when withdrawal appeared to reach its peak. However, previous research has shown that chronic ethanol vapor exposure can later enhance operant oral self-administration, even when the first opportunity to self-administer is delayed for 2 weeks after the end of vapor exposure (e.g., Roberts et al., 2000
). Future research must address whether passive IG ethanol exposure using our Massed procedure has effects on IG self-infusion that persist beyond the 12–36 h delay intervals used here.
Although the foregoing analysis suggests that the Massed procedure produced greater dependence than the Spaced procedure, it is important to note that the magnitude of withdrawal, per se, immediately or 6 h before onset of self-infusion was not a reliable predictor of ethanol intake. For example, the Massed and Spaced groups showed similar withdrawal ratings immediately before self-infusion, but showed a large difference in ethanol intake (Experiment 1). Conversely, the Immediate and Delay groups showed similar ethanol intakes, despite showing a large difference in withdrawal ratings 6 h before self-infusion (Experiment 2). Thus, although our withdrawal ratings were generally useful for ranking groups on strength of dependence, ratings made shortly before self-infusion were not useful for predicting differences in ethanol intake. One problem with comparing group ratings made just before self-infusion is that those ratings reflected different phases in the time course of withdrawal for each group. That is, the Spaced group was likely past its peak whereas the Massed group had not yet reached its peak. In fact, as shown in , the Delay-group withdrawal data (Exp. 2) suggest that rats exposed to a Massed procedure showed peak withdrawal 30 h after onset of the final passive infusion (mean score = 4.2), whereas rats exposed to the Spaced procedure (Exp. 1) showed much weaker withdrawal at that same time point (mean score = 1.3). Thus, one can speculate that the neurobiological dysregulation associated with dependence was probably greater for the Massed group than for the Spaced group during self-infusion, thereby providing a stronger basis for negative reinforcement.
The fact that Spaced group rats received more ethanol per day than Massed group rats during the last three days of the passive phase is a potential confound for interpretation of the self-infusion difference. That is, the group difference in ethanol self-infusion might be attributed to this difference in cumulative ethanol exposure rather than to differences in passive infusion “spacing” or ethanol dependence. However, this explanation seems unlikely because the expected effects of higher ethanol exposure, i.e., stronger tolerance and dependence in the Spaced group, do not readily explain the finding that the Spaced group self-infused less ethanol (see above). Our passive phase dosing procedure, which linked changes in the daily infusion dose for each rat to its own level of intoxication, was specifically designed to match groups for intoxication and to avoid overdosing. Had we attempted to match groups for dose received by increasing the dose given to Massed group rats to the level received by Spaced group rats, we would most likely have induced substantial subject attrition due to lethal overdoses in the Massed group. Alternatively, we might have reduced the daily doses given to Spaced group rats to the levels received by Massed group rats. However, in that case, interpretation of the group difference in self-infusion would be confounded by a difference in the levels of intoxication achieved during passive exposure. Moreover, there is no reason for believing that a reduction in cumulative passive ethanol exposure in the Spaced group (which would be expected to reduce tolerance and dependence) would increase later self-infusion to the levels observed in the Massed group. Because the Massed procedure produced much stronger withdrawal than the Spaced procedure, our interpretation based on the level of dependence produced by each procedure seems more plausible.
Analysis of the S+ and S− lick data and S+ preference ratios in Experiment 1 showed that Spaced Group rats developed a strong aversion for the S+ flavor whereas Massed Group rats consumed both flavors equally. Although the groups in later studies—all of which can be viewed as receiving variations of the Massed procedure—tended to show a mild aversion for S+ on choice days, their relative intakes of S+ were still generally higher than those seen in the Spaced Group or in control groups in our previous self-infusion studies (Fidler et al., 2006
). The foregoing observations raise the possibility that the Massed procedure produced greater ethanol self-infusion than the Spaced procedure because it produced greater tolerance to aversive drug effects that would otherwise condition a taste aversion to S+. The general trend toward a decrease in S+ preference ratios over days is also consistent with the suggestion that repeated pairings of the S+ flavor with IG ethanol gradually produced conditioned taste aversion.
The absence of an absolute preference for S+ over S− in our Massed IG groups contrasts with the finding of greater operant responding for oral ethanol than for water in ethanol vapor inhalation studies with rats (e.g., Funk & Koob, 2007
). Although this difference might suggest that IG self-infusion of ethanol is inherently more aversive than oral self-administration of ethanol, there is no direct evidence to support this suggestion. Moreover, our previous study showed that IG infusion (of water) by itself does not produce a conditioned aversion to a paired flavor relative to a flavor that is not paired with infusion. One important difference between previous IG and vapor inhalation studies is that the IG studies have involved continuous
(i.e., 23 h/day) access to self-infused ethanol in rats with no prior self-administration experience, whereas the vapor inhalation studies have typically involved limited
access (e.g., 0.5–12 h/day) in rats with substantial self-administration experience under limited-access conditions. It seems possible that initial training to “binge” on ethanol during limited-access sessions coupled with the use of relatively short post-exposure test sessions increased the likelihood that the vapor inhalation studies would show an absolute preference for ethanol compared to procedures like ours in which ethanol is available continuously.
In Experiment 3, we found that rats given opportunities to self-infuse ethanol on alternate days during the last few days of passive exposure (Master Group) did not alter later self-infusion compared to rats that were passively exposed to the same ethanol doses on those days (Yoked Group). We had initially hypothesized that this early experience with control over ethanol intake might result in higher self-infusion by the Masters compared to their Yoked controls, but we saw no significant differences in daily intake. In fact, there was a trend in the opposite direction, supported by the finding that a significantly higher proportion of Yoked rats (83%) than Master rats (43%) self-infused more than 5 g/kg on at least one choice day. Although rats in both groups initially received 3 consecutive days of passive ethanol exposure (massed procedure), it is possible that the interpolated opportunities to self-infuse ethanol actually interfered with further development of dependence in Master rats compared to Yoked rats, thereby offsetting any advantage provided by the earlier opportunity to self-infuse. This suggestion is supported by previous research showing that yoked exposures to ethanol (Weise-Kelly & Siegel, 2001
) or cocaine (Dworkin et al., 1995
) produce greater impairment than self-administered drug, and the hypothesis that development of (non-associative) tolerance and dependence may be enhanced in animals that experience greater drug-induced “functional impairment” (Kalant et al., 1971
The daily ethanol intakes achieved in the present studies (group means ranging from 4.1 to 7.9 g/kg/d) are notable because they generally exceeded the mean daily intakes typically reported in home cage 2-bottle choice drinking procedures for various inbred rat strains as well as the genetically heterogeneous N:NIH strain (Li & Lumeng, 1984
). Moreover, our daily intakes were similar to the daily intakes previously reported during home-cage 2-bottle choice drinking or IG self-infusion by male rats selectively bred for high ethanol intake/preference (P rats; Murphy et al., 1988
; Stewart & Li, 1997
; Waller et al., 1984
). Our daily intakes were also in the same range as those reported in a recent study that examined choice IG self-infusion procedure in Sprague-Dawley rats (5 g/kg/d using 24% v/v ethanol, Ackroff & Sclafani, 2001
). Although the terminal choice phase of that study was procedurally similar to our choice phase, the rationales, training procedures and interpretations for these two studies are quite different. Whereas those investigators were primarily interested in the ability of ethanol infusions to induce conditioned preference to a paired flavor solution, we were primarily interested in the impact of chronic ethanol exposure on ethanol self-infusion in a choice procedure. Moreover, in contrast to the relatively short period of passive exposure used in our studies (5 d), their training phase was quite lengthy (50+ d), included a period of training under food restriction (19 d) and involved extensive exposure to an ethanol concentration (6%) and daily doses that were much lower than those used in our studies. Finally, whereas our data suggest that the enhancement of ethanol intake was likely related to induction of dependence, their outcome seems better explained by ethanol’s nutritive value rather than by its pharmacological effects.
Overall, our studies add to a growing literature that shows that chronic ethanol exposure—in this case, via passive IG infusions of ethanol—can enhance subsequent self-administration of ethanol. Importantly, our data provide new information on a critical variable, namely, the periodicity of chronic ethanol exposure. Previous research using the ethanol vapor exposure procedure has shown that continuous vapor exposure is less effective than intermittent exposure for enhancing later self-administration when intermittency is defined as alternating periods of ethanol vs. no-ethanol within each 24-h period across several consecutive days of exposure (Lopez & Becker, 2005
; O’Dell et al., 2004
). Consistent with our previous studies using the IG model (Fidler et al., 2006
), the present studies also showed elevated self-administration when consecutive days of passive exposure contained alternating periods of ethanol and no-ethanol (Massed procedure). Our novel finding is that the enhancing effect of passive ethanol exposure was substantially diminished when the “off” time between periods of ethanol intoxication was extended from 12 to 36 h (Spaced Group). Based on our withdrawal data, this outcome may be best explained by proposing that the longer recovery between consecutive periods of intoxication interfered with development of dependence. When considered together with the vapor exposure data, our data suggest that the optimal schedule for passive ethanol exposure may be one that involves an ethanol-free interval of intermediate-duration (8–12 h) between periods of intoxication across consecutive days, presumably because very short (i.e., nearly continuous ethanol exposure) or very long ethanol-free intervals interfere with development of dependence.